Abstract

Nucleotide excision repair (NER) is a DNA repair pathway that processes helix distorting lesions in DNA. In humans, lesions such as UV-induced photoproducts are recognized by the UV-damaged DNA binding protein (UV-DDB). How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). Specifically, the transport mechanisms employed by UV-DDB, as well as, the stoichiometry of UV-DDB on physiologically relevant DNA substrates containing DNA damage remain unclear.To understand damage recognition by UV-DDB, we performed single molecule experiments, which revealed that the human UV-damaged DNA binding protein (UV-DDB) samples damage in DNA primarily via a three dimensional search mechanism. We found that UV-DDB displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps prior to forming long-lived, non-motile (DDB1-DDB2)2 dimers at sites of damage. To understand structure-function relationships governing DNA damage recognition by UV-DDB, we tested the xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO. We found that K244E DDB2 supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. Here we propose a framework for a conformational proofreading mechanism for specific damage recognition by UV-DDB.